The relevance of viral infections to the onset and progression of human hematologic malignancies and other blood diseases is still a matter of active investigation. Purified human T lymphocytes isolated from the peripheral blood mononuclear cells of healthy blood donors were experimentally infected with simian virus 40 (SV40), a small DNA tumor virus. SV40-positive T lymphocytes extended their lifespan up to day 80 postinfection (PI). Expression of viral antigens, such as the large T antigen and the viral capsid protein VP1 from the early and late regions, respectively, was detected up to day 40 PI. SV40 viral progeny were continuously produced from day 10 to 40 PI. SV40 DNA sequences were detected in infected T cells for up to 80 days. Our data indicate that human T lymphocytes can be efficiently infected with SV40. Although T cells infected by SV40 were not immortalized, 30% of these lymphocytes appeared to be morphologically transformed with an enlarged T-cell shape. Our investigation provides a simple model for studying the interactions of human T lymphocytes with this small DNA tumor virus and it might represent an experimental tool for investigating new biomarkers and targets for innovative therapeutic approaches.